王不留行黄酮苷改善糖尿病肾病小鼠肠道菌群紊乱和肾脏脂质沉积的研究

Study on vaccarin in improving intestinal flora disorder and renal lipid deposition in diabetes nephropathy mice

目的研究王不留行黄酮苷(Vac)对糖尿病肾病小鼠肾脏脂质合成的影响及其机制。方法通过高脂饲料喂养和腹腔注射链脲佐菌素构建糖尿病肾病小鼠模型。小鼠随机分为正常组、模型组和治疗组。采用血糖仪检测小鼠空腹血糖;采用试剂盒检测小鼠尿液中尿素氮、尿肌酐、24 h尿蛋白、尿蛋白肌酐等的含量,血清中血肌酐、甘油三酯、总胆固醇、低密度脂蛋白、高密度脂蛋白等的含量,肾脏组织中甘油三酯和总胆固醇的含量;采用HE和PAS染色法观察小鼠肾脏的病理学变化;采用总胆汁酸试剂盒测定小鼠血清中总胆汁酸的含量;通过实时荧光定量PCR、免疫印迹和免疫组化分别检测小鼠肾脏组织中固醇调节元件结合蛋白-1和脂肪酸合成酶的mRNA表达,法尼醇X受体(FXR)和G蛋白偶联胆汁酸受体5(TGR5)的mRNA和蛋白表达水平。结果与正常组比较,模型组小鼠的血糖升高、血脂增加,肾脏脂质沉积加剧,肾功能出现损伤;经Vac治疗后,糖尿病肾病小鼠可缓解脂质代谢紊乱,改善小鼠的肾脏功能。模型小鼠的肠道菌群失调,在门和属水平上的有益菌减少,有害菌增加;Vac干预可明显改善了这一情况,同时Spearman相关性分析也显示小鼠脂质代谢水平与肠道菌群存在显著相关性。与正常组比较,模型组小鼠血清中总胆汁酸的含量增加,胆汁酸受体FXR和TGR5受体的表达显著降低;Vac可降低总胆汁酸的含量,上调FXR和TGR5受体的表达。结论Vac可通过调节肠道菌群微生物及其代谢物胆汁酸的水平,缓解模型小鼠脂质沉积,可能是改善糖尿病肾病患者肾脏脂质沉积的有效候选药物。

OBJECTIVE To study the effect of vaccarin(Vac)on renal lipid synthesis in diabetes nephropathy mice and its mechanism.METHODS The diabetic nephropathy(DN)mice model was established by feeding high-fat diet and intraperitoneal injection of streptozocin.The mice were randomly divided into three groups:normal group,diabetes nephropathy model group and Vac treatment group.A glucometer was used to detect fasting blood glucose in mice.The kits were used to detect urea nitrogen,urinary creatinine,24 h urinary protein and urinary protein creatinine levels in urine,blood creatinine,triglyceride,total cholesterol,low-density lipoprotein,and high-density lipoprotein levels in serum,and triglyceride and total cholesterol levels in kidney tissue.HE and PAS staining were used to evaluate the pathological changes of mouse kidney.TBA kit was used to determine the content of total bile acid in serum of mice.The mRNA expression of sterol-regulatory element binding protein 1 and fatty acid synthase was detected by real-time fluorescence quantitative PCR.The mRNA and protein expression levels of farnesol X receptor(FXR)and G protein coupled bile acid receptor 5(TGR5)in mouse kidney tissues were detected by real-time fluorescent quantitative PCR,immunoblotting and immunohistochemistry,respectively.RESULTS Compared with the normal group,DN mice had higher blood glucose and blood lipid,increased renal lipid deposition,and impaired renal function.After treatment with Vac,Vac could alleviate lipid metabolism disorder and improve renal function in DN mice.The intestinal flora of DN mice was disordered,and the beneficial bacteria at the level of phylum and genus decreased,while the harmful bacteria increased.The intervention of Vac could significantly improve this situation.At the same time,Spearman correlation analysis also showed that the level of lipid metabolism in mice was significantly related to the intestinal flora.Compared with the normal group,the content of total bile acid in serum of the model group mice increased,and the expression of bile acid receptor FXR and TGR5 receptor decreased significantly.Vac could reduce the content of total bile acid and up regulate the expression of FXR and TGR5 receptor.CONCLUSION Vac can alleviate lipid deposition and improve renal function in DN mice by regulating the composition of intestinal flora and its metabolite bile acid.These results suggest that Vac may be an effective drug candidate to improve renal lipid deposition in patients with DN.